Imaging of LymphaticsBackgroundThe lymphatics represent the third circulation in the body and are an important route of spread for tumors. Lymphangiogenesis is a property of some tumors that allows them to propagate via the lymphatics. This process has been difficult to study because the lymphatics are small and inaccessible. However, by injecting various kinds of macromolecular contrast agents, including radiolabeled, optically labeled and magnetically labeled it is possible to identify the lymphatics and their drainage patterns(1). Moreover, the knowledge gained in this process has implications for the diagnosis and management of primary and secondary lymphedema, which can be a serious complication of cancer therapy. Finally, the development of macromolecular contrast agents has implications for other diseases.Pre-clinical researchThe MIP is investigating the role of the lymphatics in cancer using imaging methods. We have found that macromolecular agents of between 5-10nm in diameter are ideal for studying the lymphatics based on their rapid uptake and transit to the sentinel nodes. Sentinel node imaging is of importance because current methods rely on cumbersome blue dyes and radioactive sulfur colloid. We have developed a dendrimer that is dual labeled for MRI imaging and optical imaging that would allow the identification of the sentinel node without ionizing radiation using MRI and optical imaging(2). By comparing different sized dendrimers optimization of the lymphatic agent was achieved(3). We found that very small agents leaked from the lymphatics and were unsatisfactory whereas large agents were too slow. Therefore, the Generation 6 dendrimer loaded with Gadolinium and an optical fluorophore appears ideal(3-5). Working with Martin Brechbiel and Gary Griffiths we have developed and tested dendrimers with new syntheses that should simplify the chemistry for possible clinical translation(6). We have also used IgG as a carrier molecule for lymphatic imaging and shown it to be a biocompatible and suitably sized for lymphatic imaging. Furthermore, we are investigating the FDA-approved drug Ablavar for lymphatic imaging as this gadolinium chelate binds albumin and effectively becomes macromolecular. We are currently working on a dendrimer loaded with Gadolinium for potential human use. This agent uses medical grade dendrimers with a high affinity chelate, DOTA. This could be useful in the pre operative mapping of lymphatic obstruction and malformations. In addition we are exploring the role of quantum dots (QD) in identifying the lymphatics. QDs have the advantages of high quantum efficiency and very narrow emission spectra allowing multiple QDs of different wavelength to be used simultaneously(7, 8). We have employed two color QDs to investigate the drainage of the breast and arm to the axillary lymph nodes and found that some mice had independent drainage systems whereas other mice demonstrated dual draining nodes , i.e. nodes that partly drained the breast and partly drained the limb(7, 8). The implications of this anatomy for immunologic antigen processing are of potential interest. Additionally we have explored the use of up to 5 QDs of differing wavelength simultaneously in order to establish the complex lymphatic drainage patterns in the head and neck. Clinical Translation:We have been collaborating with Boston Childrens Hospital to develop a dendrimer-based MR contrast agent for use in human children with primary lymphedema. The material for experimentation in a porcine model was synthesized and will assist Boston Childrens Hospital in performing these studies. Since then we have continued to modify the agent to make it more suitable for human use. We are currently (2012) conducting safety clearance studies in rodents and larger animals using Gd-153 labeled dendrimersOne important spin-off of this work is that we have generated a Gadolinium labeled albumin molecule. Each albumin is labeled with 10-15 Gadolinium atoms. As a consequence this is an excellent imaging agent on MRI and can be used as a surrogate marker. Dr Russ Lonser (NINDS) has expressed interest in using this agent as a surrogate marker for convection enhanced delivery (CED)agents in patients with brain tumors. Current CED therapy is limited by an inability to monitor the drug. Because Gd-Albumin demonstrates similar convection properties to the agent administered for CED (IL-13 PEG), it will be a useful surrogate marker. We are also exploring the agent Ablavar which binds albumin in situ and effectively becomes a Gd-Albumin labeled product. 1.Barrett, T., Choyke, P. L., and Kobayashi, H. Imaging of the lymphatic system: new horizons. Contrast Media Mol Imaging, 1: 230-245, 2006.2.Talanov, V. S., Regino, C. A., Kobayashi, H., Bernardo, M., Choyke, P. L., and Brechbiel, M. W. Dendrimer-based nanoprobe for dual modality magnetic resonance and fluorescence imaging. Nano Lett, 6: 1459-1463, 2006.3.Kobayashi, H., Kawamoto, S., Bernardo, M., Brechbiel, M. W., Knopp, M. V., and Choyke, P. L. Delivery of gadolinium-labeled nanoparticles to the sentinel lymph node: comparison of the sentinel node visualization and estimations of intra-nodal gadolinium concentration by the magnetic resonance imaging. J Control Release, 111: 343-351, 2006.4.Koyama, Y., Talanov, V. S., Bernardo, M., Hama, Y., Regino, C. A., Brechbiel, M. W., Choyke, P. L., and Kobayashi, H. A dendrimer-based nanosized contrast agent dual-labeled for magnetic resonance and optical fluorescence imaging to localize the sentinel lymph node in mice. J Magn Reson Imaging, 25: 866-871, 2007.5.Hama, Y., Bernardo, M., Regino, C. A., Koyama, Y., Brechbiel, M. W., Krishna, M. C., Choyke, P. L., and Kobayashi, H. MR lymphangiography using dendrimer-based contrast agents: a comparison at 1.5T and 3.0T. Magn Reson Med, 57: 431-436, 2007.6.Xu, H., Regino, C. A., Bernardo, M., Koyama, Y., Kobayashi, H., Choyke, P. L., and Brechbiel, M. W. Toward improved syntheses of dendrimer-based magnetic resonance imaging contrast agents: new bifunctional diethylenetriaminepentaacetic acid ligands and nonaqueous conjugation chemistry. J Med Chem, 50: 3185-3193, 2007.7.Kobayashi, H., Hama, Y., Koyama, Y., Barrett, T., Regino, C. A., Urano, Y., and Choyke, P. L. Simultaneous multicolor imaging of five different lymphatic basins using quantum dots. Nano Lett, 7: 1711-1716, 2007.8.Hama, Y., Koyama, Y., Urano, Y., Choyke, P. L., and Kobayashi, H. Two-Color Lymphatic Mapping Using Ig-Conjugated Near Infrared Optical Probes. J Invest Dermatol, 2007.266.9. Regino CA, Walbridge S, Bernardo M, Wong KJ, Johnson D, Lonser R, Oldfield EH, Choyke PL, Brechbiel MW. A dual CT-MR dendrimer contrast agent as a surrogate marker for convection-enhanced delivery of intracerebral macromolecular therapeutic agents. Contrast Media Mol Imaging. 2008 Jan;3(1):2-8.274.10. Longmire M, Choyke PL, Kobayashi H. Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats. Nanomed. 2008 Oct;3(5):703-1711. Kobayashi H, Ogawa M, Kosaka N, Choyke PL, Urano Y. Multicolor imaging of lymphatic function with two nanomaterials: quantum dot-labeled cancer cells and dendrimer-based optical agents. Nanomed. 2009 Jun;4(4):411-9.12.Bumb A, Regino CA, Perkins MR, Bernardo M, Ogawa M, Fugger L, Choyke PL, Dobson PJ, Brechbiel MW.: Preparation and characterization of a magnetic and optical dual-modality molecular probe. Nanotechnology. 21(17):175704, 2010. 13. Kosaka N, Bernardo M, Mitsunaga M, Choyke PL, Kobayashi H.: MR and optical imaging of early micrometastases in lymph nodes: triple labeling with nano-sized agents yielding distinct signals. Contrast Media Mol Imaging. ;7(2):247-253. 2012.